Process for the fermentative production of L-amino acids by attenuation of the poxB gene

Abstract

An isolated polynucleotide containing a polynucleotide sequence selected from the group a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID no. 2, b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b) and d) polynucleotide containing at least 15 successive bases of the polynucleotide sequence of a), b) or c), and a process for the fermentative production of L-amino acids by attenuation of the poxB gene.

1 - 16 . (canceled)  
     
     
         17 . A process for the production of an amino acid, comprising the following steps: 
 a) fermentation of a bacteria producing a desired L-amino acid bacteria, in which at least the poxB gene is attenuated;    b) accumulation of the desired L-amino acid in the medium or in the cells of the bacteria; and    c) isolation of the L-amino acid.    
     
     
         18 . The process of  claim 17  wherein the amino acid is L-lysine.  
     
     
         19 . The process of  claim 17 , wherein bacteria are used in which further genes of the biosynthetic pathway of the desired L-amino acid are additionally amplified.  
     
     
         20 . The process of  claim 17 , wherein bacteria are used in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partially suppressed.  
     
     
         21 . The process of  claim 17 , wherein expression of a polynucleotide containing a polynucleotide sequence selected from the group consisting of 
 a) a polynucleotide which is at least 70% identical to a polynucleotide which encodes a polypeptide containing the amino acid sequence of SEQ ID NO:2,    b) a polynucleotide which encodes a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2,    c) a polynucleotide which is complementary to the polynucleotides of a) or b), and    d) a polynucleotide containing at least 15 successive bases of the polynucleotide sequence of a), b) or c), is reduced.    
     
     
         22 . The process of  claim 17 , wherein the catalytic properties of a polypeptide containing a polynucleotide sequence selected from the group consisting of 
 a) a polynucleotide which is at least 70% identical to a polynucleotide which encodes a polypeptide containing the amino acid sequence of SEQ ID NO:2,    b) a polynucleotide which encodes a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2,    c) a polynucleotide which is complementary to the polynucleotides of a) or b), and    d) a polynucleotide containing at least 15 successive bases of the polynucleotide sequence of a), b) or c), are reduced.    
     
     
         23 . The process of  claim 17 , wherein bacteria are used in which attenuation is achieved by using integration mutagenesis by means of the plasmid pCR2.1poxbint, shown in  FIG. 1  and deposited as DSM 13114, or one of the constituents thereof.  
     
     
         24 . The process of  claim 17 , wherein bacteria of the genus  Corynebacterium glutamicum  are used.  
     
     
         25 . A process for the fermentative preparation of L-lysine in  Corynebacterium glutamicum  comprising: 
 (a) growing said  Corynebacterium glutamicum  in which a polynucleotide encoding a PoxB polypeptide of SEQ ID NO: 2 is attenuated by a method of mutagenesis selected from the group consisting of insertion mutagenesis by insertion of at least one base pair, deletion mutagenesis with deletion of at least one base pair, and transition or transversion mutagenesis with incorporation of a non-sense mutation or the activity of said polypeptide is reduced as compared to an unattenuated  Corynebacterium glutamicum;      (b) concentrating the L-lysine in the medium or  Corynebacterium glutamicum  cells; and    (c) isolating said L-amino acid product.    
     
     
         26 . The process of  claim 25 , wherein said polynucleotide is attenuated by integration mutagenesis using plasmid pCR2.1poxBint, as shown in  FIG. 1 , and deposited as DSM 13114.  
     
     
         27 . The process of  claim 25 , further comprising fermenting  C. glutamicum  in which one or more genes selected from the group consisting of: 
 (a) a dapA gene which codes for dihydrodipicolinate synthase;    (b) a pyc gene which encodes pyruvate carboxylase;    (c) a dapE gene which encodes succinyldiaminopimelate desuccinylase;    (d) a dap gene which encodes glyceraldehyde 3-phosphate dehydrogenase;    (e) a mqo gene which encodes a malate:quinone oxidoreductase; and    (f) a lysE gene which encodes a lysine export protein,    are simultaneously over-expressed by increasing the copy number or operatively linking to a heterologous promoter.    
     
     
         28 . A process for the fermentative preparation of L-lysine in  Corynebacterium  comprising: 
 (a) growing said  Corynebacterium  in which a  Corynebacterium  poxB gene is attenuated by a method of mutagenesis selected from the group consisting of insertion mutagenesis by insertion of at least one base pair, deletion mutagenesis with deletion of at least one base pair, and transition or transversion mutagenesis with incorporation of a non-sense mutation or the activity of said polypeptide is reduced as compared to an unattenuated  Corynebacterium;      (b) concentrating the L-lysine in the medium or  Corynebacterium  cells; and    (c) isolating said L-amino acid product.    
     
     
         29 . The process of  claim 28 , wherein said polynucleotide is attenuated by integration mutagenesis using plasmid pCR2.1poxBint, as shown in  FIG. 1 , and deposited as DSM 13114.  
     
     
         30 . The process of  claim 29 , further comprising fermenting  Corynebacterium  in which one or more genes selected from the group consisting of: 
 (a) a dapA gene which codes for dihydrodipicolinate synthase;    (b) a pyc gene which encodes pyruvate carboxylase;    (c) a dapE gene which encodes succinyldiaminopimelate desuccinylase;    (d) a dap gene which encodes glyceraldehyde 3-phosphate dehydrogenase;    (e) a mqo gene which encodes a malate:quinone oxidoreductase; and    (f) a lysE gene which encodes a lysine export protein,    are simultaneously over-expressed by increasing the copy number or operatively linking to a heterologous promoter.